The present invention is directed to a refined detoxified endotoxin (RDE) product which, when combined with cell wall skeleton (CWS), results in a therapeutically effective composition for the treatment of cancerous tumors without the deleterious side effects normally associated with endotoxins. The RDE used in the present composition is characterized as having no detectable 2-keto-3-deoxyoctanoate, between about 350 and 475 nmoles/mg of phosphorus and between about 1700 and 2000 nmoles/mg of fatty acids. The present invention also includes methods of preparing RDE as well as the use of the RDE-CWS composition to obtain regression and remission of cancerous tumors.
Endotoxic extracts obtained from Enterobacteriaciae including parent organisms and mutants are known. These extracts have been used for immunotherapy of various immunogenic tumors [see Peptides as Requirement for Immunotherapy of the Guinea-Pig Line-10 Tumor with Endotoxins; Ribi, et al Cancer Immunol. Immunother., Vol. 7, pgs. 43-58 (1979) incorporated herein by reference]. However, the endotoxin extracts are known to be highly toxic and, therefore, of limited use in the treatment of cancerous tumors. Efforts have been made to "detoxify" the endotoxins while retaining their tumor regressive capacity. As shown, in Ribi, et al, chemical procedures known to "detoxify" endotoxins while retaining adjuvanticity, such as succinylation and phthalylation resulted in both loss of endotoxicity and tumor regressive potency. Therefore, prior art attempts to obtain an endotoxin product with high tumor regressive potency and little or no toxicity have thus far not been successful.
Cell wall skeleton is essentially cell wall which has had much of the protein and lipids normally found in the cell wall removed. It is a polymeric mycolic acid arabinogalactan mucopeptide containing remnants of trehalose mycolate ("P.sub.3 ") and undigested tuberculoproteins. Cell wall skeleton is obtained from any mycobacteria including, but not limited to, M.smegmatis, M.phlei, Nocardia rubra, Nocardia asteroides, Corynebacterium diphtheriae, Corynebacterium parvum, M.kansasii, M.tuberculosis (Strain H 37 RV and Ayoma B), and M.bovis, Strain BCG. Additionally, cell wall skeleton may be obtained from such non-mycobacteria as E.coli, B.abortus and Coxiella burnettii.
Cell wall skeleton is produced by first growing and harvesting a bacteria such as M.bovis, Strain BCG (bacillus Calmette-Guerin). The resulting whole cell residue is processed through a cell fractionator [Ribi Cell Fractionator (Sorvall, Model RF-1)] which disrupts the cells separating the outer envelope or cell wall from the protoplasmic impurities. The resulting cell walls are then subjected to a series of solvent extractions and enzymatic treatments (e.g. trypsin and/or chymotrypsin) to give purified cell wall skeleton.
It is, therefore, an object of the present invention to produce a superior refined detoxified endotoxin product which has a high tumor regressive potency without toxic side effects normally associated therewith. It is another object of the present invention to provide a refined detoxified endotoxin in combination with cell wall skeleton as a highly potent composition for the treatment of cancerous tumors.